Variant forms of urate oxidase and use thereof

ABSTRACT

Genetically modified proteins with uricolytic activity are described. Proteins comprising truncated urate oxidases and methods for producing them, including PEGylated proteins comprising truncated urate oxidase are described.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.13/461,170 filed May 1, 2012, now allowed, which is a divisionalapplication of U.S. application Ser. No. 11/918,297 filed Dec. 11, 2008,now U.S. Pat. No. 8,188,224, which is a national stage filing ofcorresponding international application number PCT/US2006/013660, filedon Apr. 11, 2006, which claims priority to and benefit of U.S.provisional application Ser. No. 60/670,573, filed on Apr. 11, 2005, thedisclosure of which is being incorporated by reference herein.

FIELD OF INVENTION

The present invention relates to genetically modified proteins withuricolytic activity. More specifically, the invention relates toproteins comprising truncated urate oxidases and methods for producingthem.

BACKGROUND OF THE INVENTION

The terms urate oxidase and uricase are used herein interchangeably.Urate oxidases (uricases; E.C. 1.7.3.3) are enzymes which catalyze theoxidation of uric acid to a more soluble product, allantoin, a purinemetabolite that is more readily excreted. Humans do not produceenzymatically active uricase, as a result of several mutations in thegene for uricase acquired during the evolution of higher primates. Wu,X, et al., (1992) J Mol Evol 34:78-84, incorporated herein by referencein its entirety. As a consequence, in susceptible individuals, excessiveconcentrations of uric acid in the blood (hyperuricemia) can lead topainful arthritis (gout), disfiguring urate deposits (tophi) and renalfailure. In some affected individuals, available drugs such asallopurinol (an inhibitor of uric acid synthesis) producetreatment-limiting adverse effects or do not relieve these conditionsadequately. Hande, K R, et al., (1984) Am J Med 76:47-56; Fam, A G,(1990) Bailliere's Clin Rheumatol 4:177-192, each incorporated herein byreference in its entirety. Injections of uricase can decreasehyperuricemia and hyperuricosuria, at least transiently. Since uricaseis a foreign protein in humans, even the first injection of theunmodified protein from Aspergillus flavus has induced anaphylacticreactions in several percent of treated patients (Pui, C-H, et al.,(1997) Leukemia 11:1813-1816, incorporated herein by reference in itsentirety), and immunologic responses limit its utility for chronic orintermittent treatment. Donadio, D, et al., (1981) Nouv Presse Med10:711-712; Leaustic, M, et al., (1983) Rev Rhum Mal Osteoartic50:553-554, each incorporated herein by reference in its entirety.

The sub-optimal performance of available treatments fur hyperuricemiahas been recognized for several decades. Kissel, P, et al., (1968)Nature 217:72-74, incorporated herein by reference in its entirety.Similarly, the possibility that certain groups of patients with severegout might benefit from a safe and effective form of injectable uricasehas been recognized for many years. Davis, F F., et al., (1978) in G BBroun, et al., (Eds.) Enzyme Engineering, Vol. 4 (pp. 169-173) New York,Plenum Press; Nishimura, H, et al., (1979) Enzyme 24:261-264; Nishimura,H, et al., (1981) Enzyme 26:49-53; Davis, S, et al., (1981) Lancet2(8241):281-283; Abuchowski, A, et al., (1981) J Pharmacol Exp Ther219:352-354; Chen, R H-L, et al., (1981) Biochim Biophys Acta660:293-298; Chua, C C, et al., (1988) Ann Int Med 109:114-117;Greenberg, M L, et al., (1989) Anal Biochem 176:290-293, eachincorporated herein by reference in its entirety. Uricases derived fromanimal organs are nearly insoluble in solvents that are compatible withsafe administration by injection. U.S. Pat. No. 3,616,231, incorporatedherein by reference in its entirety. Certain uricases derived fromplants or from microorganisms are more soluble in medically acceptablesolvents. However, injection of the microbial enzymes quickly inducesimmunological responses that can lead to life-threatening allergicreactions or to inactivation andior accelerated clearance of the uricasefrom the circulation. Donadio, et al., (1981); Leaustic, et al., (1983).Enzymes based on the deduced amino acid sequences of uricases frommammals, including pig and baboon, or from insects, such as, forexample, Drosophila melanogaster or Drosophila pseudoobscura (Wallrath,L L, et al., (1990) Mol Cell Biol 10:5114-5127, incorporated herein byreference in its entirety), have not been suitable candidates forclinical use, due to problems of immunogenicity and insolubility atphysiological pH.

Previously, investigators have used injected uricase to catalyze theconversion of uric acid to allantoin in vivo. See Pui, et al., (1997).This is the basis for the use in France and Italy of uricase from thefungus Aspergillus flavus (URICOZYME®) to prevent or temporarily correctthe hyperuricemia associated with cytotoxic therapy for hematologicmalignancies and to transiently reduce severe hyperuricemia in patientswith gout. Potaux, L, et al., (1975) Nouv Presse Med 4:1109-1112;Legoux, R, et al., (1992) J biol Chem 267:8565-8570; U.S. Pat. Nos.5,382,518 and 5,541,098, each incorporated herein by reference in itsentirety. Because of its short circulating lifetime, URICOZYME® requiresdaily injections. Furthermore, it is not well suited for long-termtherapy because of its immunogenicity.

Certain uricases are useful for preparing conjugates with poly(ethyleneglycol) or poly(ethylene oxide) (both referred to as PEG) to producetherapeutically efficacious forms of uricase having increased proteinhalf-life and reduced immunogenicity. U.S. Pat. Nos. 4,179,337,4,766,106, 4,847,325, and 6,576,235; U.S. Patent Application PublicationUS2003/0082786A1, each incorporated herein by reference in its entirety.Conjugates of uricase with polymers other than PEG have also beendescribed. U.S. Pat. No. 4,460,683, incorporated herein by reference inits entirety.

In nearly all of the reported attempts to PEGylate uricase (i.e. tocovalently couple PEG to uricase), the PEG is attached primarily toamino groups, including the amino-terminal residue and the availablelysine residues. In the uricases commonly used, the total number oflysines in each of the four identical subunits is between 25(Aspergillus flavus (U.S. Pat. No. 5,382,518, incorporated herein byreference in its entirety)) and 29 (pig (Wu, X, et al., (1989) Proc NatlAcad Sci USA 86:9412-9416, incorporated herein by reference in itsentirety)). Some of the lysines are unavailable for PEGylation in thenative conformation of the enzyme. The most common approach to reducingthe immunogenicity of uricase has been to couple large numbers ofstrands of low molecular weight PEG. This has invariably resulted inlarge decreases in the enzymatic activity of the resultant conjugates.

A single intravenous injection of a preparation of Candida itilisuricase coupled to 5 kDa PEG reduced serum urate to undetectable levelsin five human subjects whose average pre-injection serum urateconcentration is 6.2 mg/dl, which is within the normal range. Davis, etal., (1981). The subjects were given an additional injection four weekslater, but their responses were not reported. No antibodies to uricasewere detected following the second (and last) injection, using arelatively insensitive gel diffusion assay. This reference reported noresults from chronic or subchronic treatments of human patients orexperimental animals.

A preparation of uricase from Arthrobacter protoformiae coupled to 5 kDaPEG was used to temporarily control hyperuricemia in a single patientwith lymphoma whose pre-injection serum urate concentration is 15 mg/dL.Chua, et al., (1988). Because of the critical condition of the patientand the short duration of treatment (four injections during 14 days), itis not possible to evaluate the long-term efficacy or safety of theconjugate.

Improved protection from immune recognition is enabled by modifying eachuricase subunit with 2-10 strands of high molecular weight PEG (>5kD-120 kD) Saifer, et al. (U.S. Pat. No. 6,576,235; (1994) Adv Exp MedBiol 366:377-387, each incorporated herein by reference in itsentirety). This strategy enabled retention of >75% enzymatic activity ofuricase from various species, following PEGylation, enhanced thecirculating life of uricase, and enabled repeated injection of theenzyme without eliciting antibodies in mice and rabbits.

Hershfield and Kelly (International Patent Publication WO 00/08196; U.S.Application No. 60/095,489, incorporated herein by reference in itsentirety) developed means for providing recombinant uricase proteins ofmammalian species with optimal numbers of PEGylation sites. They used PMtechniques to increase the number of available lysine residues atselected points on the enzyme which is designed to enable reducedrecognition by the immune system, after subsequent PEGylation, whilesubstantially retaining the enzyme's uricolytic activity. Some of theiruricase proteins are truncated at the carboxy and/or amino termini. Theydo not provide for directing other specific genetically-inducedalterations in the protein.

In this application, the term “immunogenicity” refers to the inductionof an immune response by an injected preparation of PEG-modified orunmodified uricase (the antigen), while “antigenicity” refers to thereaction of an antigen with preexisting antibodies. Collectively,antigenicity and immunogenicity are referred to as “immunoreactivity.”In previous studies of PEG-uricase, immunoreactivity is assessed by avariety of methods, including: 1) the reaction in vitro of PEG-uricasewith preformed antibodies; 2) measurements of induced antibodysynthesis; and 3) accelerated clearance rates after repeated injections.

Previous attempts to eliminate the immunogenicity of uricases fromseveral sources by coupling various numbers of strands of PEG throughvarious linkers have met with limited success. PEG-uricases were firstdisclosed by F F Davis and by V Inada and their colleagues. Davis, etal., (1978); U.S. Pat. No. 4,179,337; Nishimura, et al., (1979);Japanese Patents 55-99189 and 62-55079, each incorporated herein byreference in its entirety. The conjugate disclosed in U.S. Pat. No.4,179,337 is synthesized by reacting uricase of unspecified origin witha 2,000-fold molar excess of 750 dalton PEG, indicating that a largenumber of polymer molecules is likely to have been attached to eachuricase subunit. U.S. Pat. No. 4,179,337 discloses the coupling ofeither PEG or poly(propylene glycol) with molecular weights of 500 to20,000 daltons, preferably about 500 to 5,000 daltons, to provideactive, water-soluble, non-immunogenic conjugates of various polypeptidehormones and enzymes including oxidoreductases, of which uricase is oneof three examples. In addition, U.S. Pat. No. 4,179,337 emphasizes thecoupling of 10 to 100 polymer strands per molecule of enzyme, and theretention of at least 40% of enzymatic activity. No test results werereported for the extent of coupling of PEG to the available amino groupsof uricase, the residual specific uricolytic activity, or theimmunoreactivity of the conjugate.

In previous publications, significant decreases in uricolytic activitymeasured in vitro were caused by coupling various numbers of strands ofPEG to uricase from Candida utilis. Coupling a large number of strandsof 5 kDa PEG to porcine liver uricase gave similar results, as describedin both the Chen publication and a symposium report by the same group.Chen, et al., (1981); Davis, et al., (1978).

In seven previous studies, the immunoreactivity of uricase is reportedto be decreased by PEGylation and was eliminated in five other studies.In three of the latter five studies, the elimination of immunoreactivityis associated with profound decreases in uricolytic activityto at most15%, 28%, or 45% of the initial activity. Nishimura, et al., (1979) (15%activity); Chen, et al., (1981) (28% activity); Nishimura, et al.,(1981) (45% activity). In the fourth report, PEG is reported to becoupled to 61% of the available lysine residues, but the residualspecific activity is not stated. Abuchowski, et al., (1981). However, aresearch team that included two of the same scientists and used the samemethods reported elsewhere that this extent of coupling left residualactivity of only 23-28%. Chen, et al., (1981). The 1981 publications ofAbuchowski et al., and Chen et al., indicate that to reduce theimmunogenicity of uricase substantially, PEG must be coupled toapproximately 60% of the available lysine residues. The fifthpublication in which the immunoreactivity of uricase is reported to havebeen eliminated does not disclose the extent of PEG coupling, theresidual uricolytic activity, or the nature of the PEG-protein linkage.Veronese, F M, et al., (1997) in J M Harris, et al., (Eds.),Poly(ethylene glycol) Chemistry and Biological Applications. ACSSymposium Series 680 (pp. 182-192) Washington, D.C.: American ChemicalSociety, incorporated herein by reference in its entirety.

Conjugation of PEG to a smaller fraction of the lysine residues inuricase reduced but did not eliminate its immunoreactivity inexperimental animals. Tsuji, J, et al., (1985) Int J Immunopharmacol7:725-730, incorporated herein by reference in its entirety (28-45% ofthe amino groups coupled); Yasuda, Y, et al., (1990) Chem Pharm Bull38:2053-2056, incorporated herein by reference in its entirety (38% ofthe amino groups coupled). The residual uricolytic activities of thecorresponding adducts ranged from <33% (Tsuji, et al.) to 60% (Yasuda,et al.) of their initial values. Tsuii, et al., synthesized PEG-uricaseconjugates with 7.5 kDa and 10 kDa PEGs, in addition to 5 kDa PEG. Allof the resultant conjugates are somewhat immunogenic and antigenic,while displaying markedly reduced enzymatic activities.

A PEGylated preparation of uricase from Candida utilis that is safelyadministered twice to each of five humans is reported to have retainedonly 11% of its initial activity. Davis, et al., (1981). Several yearslater, PEG-modified uricase from Arthrobacter protoformiae wasadministered four times to one patient with advanced lymphoma and severehyperuricemia. Chua, et al., (1988). While the residual activity of thatenzyme preparation was not measured, Chua, et al., demonstrated theabsence of anti-uricase antibodies in the patient's serum 26 days afterthe first PEG-uricase injection, using an enzyme-linked immunosorbentassay (ELISA).

Previous studies of PEGylated uricase show that catalytic activity ismarkedly depressed by coupling a sufficient number of strands of PEG todecrease its immunoreactivity substantially. Furthermore, most previouspreparations of PEG-uricase are synthesized using PEG activated withcyanuric chloride, a triazine derivative(2,4,6-trichloro-1,3,5-triazine) that has been shown to introduce newantigenic determinants and to induce the formation of antibodies inrabbits. Tsuji, et al., (1985).

Japanese Patent No. 3-148298 to A Sano, et al., incorporated herein byreference in its entirety, discloses modified proteins, includinguricase, derivatized with PEG having a molecular weight of 1-12 kDa thatshow reduced antigenicity and “improved prolonged” action, and methodsof making such derivatized peptides. However, there are no disclosuresregarding strand counts, enzyme assays, biological tests or the meaningof “improved prolonged.” Japanese Patents 55-99189 and 62-55079, eachincorporated herein by reference in its entirety, both to Y Inada,disclose uricase conjugates prepared with PEG-triazine orbis-PEG-triazine (denoted as PEG₂), respectively. See Nishimura, et al.,(1979 and 1981). In the first type of conjugate, the molecular weightsof the PEGs are 2 kDa and 5 kDa, while in the second, only 5 kDa PEG isused. Nishimura, et al., (1979) reported the recovery of 15% of theuricolytic activity after modification of 43% of the available lysineswith linear 5 kDa PEG, while Nishimura, et al., (1981) reported therecovery of 31% or 45% of the uricolytic activity after modification of46% or 36% of the lysines, respectively, with PEG₂.

Previously studied uricase proteins were either natural or recombinantproteins. However, studies using SDS-PAGE and/or Western techniquesrevealed the presence of unexpected low molecular weight peptides whichappear to be degradation products and increase in frequency over time.The present invention is related to mutant recombinant uricase proteinshaving truncations and enhanced structural stability.

SUMMARY OF THE INVENTION

The present invention provides novel recombinant uricase proteins. Inone embodiment, the proteins of the invention contemplated are truncatedand have mutated amino acids relative to naturally occurring uricaseproteins. In particular embodiments, the mutations are at or around theareas of amino acids 7, 46, 291, and 301. Conservative mutationsanywhere in the peptide are also contemplated as a part of theinvention.

The subject invention provides a mutant recombinant uricase, wherein theuricase has been truncated by 1-20 amino acids and retains theuricolytic activity of the naturally occurring uricase. The truncationsare at or around the sequence termini such that the protein may containthe ultimate amino acids. These mutations and truncations may enhancestability of the protein comprising such mutations.

In another embodiment, the present invention to provides a means formetabolizing uric acid comprising a novel recombinant uricase proteinhaving uricolytic activity. Uricolytic activity is used herein to referto the enzymatic conversion of uric acid to allantoin.

The subject invention further provides a host cell with the capacity forproducing a uricase that has been truncated by 1-20 amino acids, and hasmutated amino acids and retains uricolytic activity.

In an embodiment, an isolated truncated mammalian uricase is providedcomprising a mammalian uricase amino acid sequence truncated at theamino terminus or the carboxy terminus or both the amino and carboxytermini by about 1-13 amino acids and further comprising an amino acidsubstitution at about position 46. In particular embodiments, theuricase comprises an amino terminal amino acid, wherein the aminoterminal amino acid is alanine, glycine, proline, serine, or threonine.Also provided is a uricase wherein there is a substitution at aboutposition 46 with threonine or alanine. In an embodiment, the uricasecomprises the amino acid sequence of SEQ II) NO. 8. In an embodiment,the uricase is conjugated with a polymer to form, for example, apolyethylene glycol-uricase conjugate. In particular embodiments,polyethylene glycol-uricase conjugates comprise 2 to 12 polyethyleneglycol molecules on each uricase subunit, preferably 3 to 10polyethylene glycol molecules per uricase subunit. In particularembodiments, each polyethylene glycol molecule of the polyethyleneglycol-uricase conjugate has a molecular weight between about 1 kD and100 kD; about 1 kD and 50 kD; about 5 kD and 20 kD; or about 10 kD. Alsoprovided are pharmaceutical compositions comprising the uricase of theinvention, including the polyethylene glycol-uricase conjugate. In anembodiment, the pharmaceutical composition is suitable for repeatedadministration.

Also provided is a method of reducing uric acid levels in a biologicalfluid of a subject in need thereof, comprising administering thepharmaceutical composition comprising the uricase of the invention. In aparticular embodiment, the biological fluid is blood.

In an embodiment, the uricase comprises a peptide having the sequence ofposition 44 to position 56 of Pig-KS-ΔN (SEQ ID NO. 14).

In an embodiment, the uricase protein comprises an N-terminalmethionine. In a particular embodiment, the uricase comprises the aminoacid sequence of SEQ ID NO. 7.

Also provided are isolated nucleic acids comprising a nucleic acidsequence which encodes a uricase of the invention, for example, uricaseshaving or comprising the amino acid sequences of SEQ ID NO. 7, SEQ IDNO. 8, SEQ ID NO. 12 or SEQ ID NO. 13. In an embodiment, the isolatednucleic acid is operatively linked to a heterologous promoter, forexample, the osmB promoter. Also provided are vectors comprising uricaseencoding nucleic acids, and host cells comprising such vectors. In anembodiment, the nucleic acid has the sequence of SEQ ID NO. 7. Alsoprovided is a method for producing a uricase comprising the steps ofculturing such a host cell under conditions such that uricase isexpressed by the host cell and isolating the expressed uricase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the structure of plasmid pOUR-P-ΔN-ks-1. Numbers nextto restriction sites indicate nucleotide position, relative to HaeIIsite, designated as 1. Restriction sites which are lost during cloningare marked in parenthesis.

FIG. 2 depicts the DNA and the deduced amino acid sequences of Pig-KS-ΔNuricase (SEQ ID NO. 9 and SEQ ID NO. 7, respectively). The amino acidnumbering in FIG. 2 is relative to the complete pig uricase sequence.Following the initiator methionine residue, a threonine replacesaspartic acid 7 of the pig uricase sequence. The restriction sites thatare used for the various steps of subcloning are indicated. The 3′untranslated sequence is shown in lowercase letters. The translationstop codon is indicated by an asterisk.

FIG. 3 shows relative alignment of the deduced amino acid sequences ofthe various recombinant pig (SEQ ID NO. 11), PBC-ΔNC (SEQ ID NO. 12),and Pig-KS-ΔN (SEQ ID NO. 7) uricase sequences. The asterisks indicatethe positions in which there are differences in amino acids in thePig-KS-ΔN as compared to the published pig uricase sequence; the circlesindicate positions in which there are differences in amino acids inPig-KS-ΔN as compared to PBC-ΔN. Dashed lines indicate deletion of aminoacids.

FIG. 4 depicts SDS-PAGE of pig uricase and the highly purified uricasevariants produced according to Examples 1-3. The production date(month/year) and the relevant lane number for each sample is indicatedin the key below. The Y axis is labeled with the weights of molecularweight markers, and the top of the figure is labeled with the lanenumbers. The lanes are as follows: Lane 1—Molecular weight markers; Lane2—Pig KS-ΔN (7/98); Lane 3—Pig (9/98); Lane 4—Pig KS (6/99); Lane 5—PigKS (6/99); Lane 6—Pig-A (6/99); Lane 7—Pig KS-ΔN (7/99); Lane 8—PigKS-ΔN (8/99).

FIG. 5 depicts the pharmacokinetic profiles of PEGylated (9×10 kD)Pig-KS-ΔN uricase in rats following IM (intramuscular), SC(subcutaneous), and IV (intravenous) injections, as determined bymonitoring enzymatic activity in blood samples. Uricase activity inplasma samples, which are collected at the indicated time points, isdetermined using the colorimetric assay. Activity values(mAti=milli-absorbance units) represent the rate of enzymatic reactionper 1 μl of plasma sample. The bioavailability (amount of drug reachingthe circulation relative to an IV injection) of uricase injected wascalculated from the area under the curve of the graph.

FIG. 6 depicts the pharmacokinetic profiles of PEGylated (9×10 kD)Pig-KS-ΔN uricase in rabbits following IM (intramuscular), SC(subcutaneous), and IV (intravenous) injections, as determined bymonitoring enzymatic activity in blood samples. Uricase activity inplasma samples collected at the indicated time points is determinedusing a colorimetric assay. Activity values (mALT=milli-absorbanceunits) represent the rate of enzymatic reaction per 1 μl of plasmasample. The bioavailability (amount of drug reaching the circulationrelative to an IV injection) of uricase injected was calculated from thearea under the curve of the graph.

FIG. 7 depicts the pharmacokinetic profiles of PEGylated (9×10 kD)Pig-KS-ΔN uricase in dogs following IM (intramuscular), SC(subcutaneous), and IV (intravenous) injections, as determined bymonitoring enzymatic activity in blood samples. Uricase activity inplasma samples, which are collected at the indicated time points, isdetermined using the calorimetric assay. Activity values(mAti=milli-absorbance units) represent the rate of enzymatic reactionper 1 μl of plasma sample. The bioavailability (amount of drug reachingthe circulation relative to an IV injection) of uricase injected wascalculated from the area under the curve of the graph.

FIG. 8 depicts the pharmacokinetic profiles of PEGylated (9×10 kD)Pig-KS-ΔN uricase in pigs following IM (intramuscular), SC(subcutaneous), and IV (intravenous) injections, as determined bymonitoring enzymatic activity in blood samples. Uricase activity inplasma samples, which are collected at the indicated time points, isdetermined using the colorimetric assay. Activity values(mAU=milli-absorbance units) represent the rate of enzymatic reactionper 1 μl of plasma sample. The bioavailability (amount of drug reachingthe circulation relative to an IV injection) of uricase injected wascalculated from the area under the curve of the graph.

DETAILED DESCRIPTION OF THE INVENTION

Previous studies teach that when a significant reduction in theimmunogenicity and/or antigenicity of uricase is achieved by PEGylation,it is invariably associated with a substantial loss of uricolyticactivity. The safety, convenience and cost-effectiveness ofbiopharmaceuticals are all adversely impacted by decreases in theirpotencies and the resultant need to increase the administered dose.Thus, there is a need for a safe and effective alternative means forlowering elevated levels of uric acid in body fluids, including blood.The present invention provides a mutant recombinant uricase, wherein theuricase has been truncated by 1-20 amino acids at either the aminoterminus or the carboxy terminus, or both, and substantially retainsuricolytic activity of the naturally occurring uricase.

Uricase, as used herein, includes individual subunits, as well as thetetramer, unless otherwise indicated.

Mutated uricase, as used herein, refers to uricase molecules havingamino acids exchanged with other amino acids.

A conservative mutation, as used herein, is a mutation of one or moreamino acids, at or around a position, that does not substantially alterthe protein's behavior. In a preferred embodiment, the uricasecomprising at least one conservative mutation has the same uricaseactivity as does uricase without such mutation. In alternateembodiments, the uricase comprising at least one conservative mutationhas substantially the same uricase activity, within 5% of the activity,within 10% of the activity, or within 30% of the activity of uricasewithout such mutation.

Conservative amino acid substitution is defined as a change in the aminoacid composition by way of changing amino acids of a peptide,polypeptide or protein, or fragment thereof. In particular embodiments,the uricase has one, two, three or four conservative mutations. Thesubstitution is of amino acids with generally similar properties (e.g.,acidic, basic, aromatic, size, positively or negatively charged, polar,non-polar) such that the substitutions do not substantially alterpeptide, polypeptide or protein characteristics (e.g., charge, IEF,affinity, avidity, conformation, solubility) or activity. Typicalsubstitutions that may be performed for such conservative amino acidsubstitution may be among the groups of amino acids as follows:

glycine (G), alanine (A), valine (V), leucine (IL) and isoleucine (I)

aspartic acid (D) and glutamic acid (E)

alanine (A), serine (S) and threonine (T)

histidine (H), lysine (K) and arginine (R)

asparagine (N) and glutamine (Q)

phenylalanine (F), tyrosine (Y) and tryptophan (W)

The protein having one or more conservative substitutions retains itsstructural stability and can catalyze a reaction even though its DNAsequence is not the same as that of the original protein.

Truncated uricase, as used herein, refers to uricase molecules havingshortened primary amino acid sequences. Amongst the possible truncationsare truncations at or around the amino and/or carboxy termini. Specifictruncations of this type may be such that the ultimate amino acids(those of the amino and/or carboxy terminus) of the naturally occurringprotein are present in the truncated protein. Amino terminal truncationsmay begin at position 1, 2, 3, 4, 5 or 6. Preferably, the amino terminaltruncations begin at position 2, thereby leaving the amino terminalmethionine. This methionine may be removed by post-translationalmodification. In particular embodiments, the amino terminal methionineis removed after the uricase is produced. In a particular embodiment,the methionine is removed by endogenous bacterial aminopeptidase.

A truncated uricase, with respect to the full length sequence, has oneor more amino acid sequences excluded. A protein comprising a truncateduricase may include any amino acid sequence in addition to the truncateduricase sequence, but does not include a protein comprising a uricasesequence containing any additional sequential wild type amino acidsequence. In other words, a protein comprising a truncated uricasewherein the truncation begins at position 6 (i.e., the truncated uricasebegins at position 7) does not have, immediately upstream from thetruncated uricase, whatever amino acid that the wild type uricase has atposition 6.

Unless otherwise indicated by specific reference to another sequence ora particular SEQ ID NO., reference to the numbered positions of theamino acids of the uricases described herein is made with respect to thenumbering of the amino acids of the pig uricase sequence. The amino acidsequence of pig uricase and the numbered positions of the amino acidscomprising that sequence may be found in FIG. 3. As used herein,reference to amino acids or nucleic acids “from position X to positionY” means the contiguous sequence beginning at position X and ending atposition Y, including the amino acids or nucleic acids at both positionsX and Y.

Uricase genes and proteins have been identified in several mammalianspecies, for example, pig, baboon, rat, rabbit, mouse, and rhesusmonkey. The sequences of various uricase proteins are described hereinby reference to their public data base accession numbers, as follows:gi|50403728|sp|P25689; gi|20513634|dbj|BAB91555.1; gi|176610|AAA35395.1;gi|205136541dbj|BAB91557.1; gi|47523606|ref|NP_999435.1;gi|6678509|ref|NP_033500.1; gi|57463|emb|CAA31490.1;gi|20127395|ref|NP_446220.1; gi|137107|sp|P11645;gi|5145866|ref|XP_497688. 1; gi|207619|gb|AAA42318.1;gi|26340770|dbj|BAC34047.1; and gi|57459|emb|CAA30378.1. Each of thesesequences and their annotations in the public databases accessiblethrough the National Center for Biotechnology Information (NCBI) isincorporated by reference in its entirety.

In an embodiment of the invention, the uricase is truncated by 4-13amino acids at its amino terminus. In an embodiment of the invention,the uricase is truncated by 4-13 amino acids at its carboxy terminus. Inan embodiment of the invention, the uricase is truncated by 4-13 aminoacids at both its carboxy and amino termini.

In an embodiment of the invention, the uricase is truncated by 6 aminoacids at its amino terminus. In an embodiment of the invention, theuricase is truncated by 6 amino acids at its carboxy terminus. In anembodiment of the invention, the uricase is truncated by 6 amino acidsat both its carboxy and amino termini.

In a particular embodiment, the uricase protein comprises the amino acidsequence from position 13 to position 292 of the amino acid sequence ofpig uricase (SEQ ID NO. 11). In a particular embodiment, the uricaseprotein comprises the amino acid sequence from position 8 to position287 of the amino acid sequence of PBC-ΔNC (SEQ ID NO. 12). In aparticular embodiment, the uricase protein comprises the amino acidsequence from position 8 to position 287 of the amino acid sequence ofPig-KS-ΔN (SEQ ID NO. 7).

In another embodiment, the uricase protein comprises the amino acidsequence from position 44 to position 56 of Pig-KS-ΔN (SEQ ID NO. 14).This region of uricase has homology to sequences within the tunnelingfold (T-fold) domain of uricase, and has within it a mutation atposition 46 with respect to the native pig uricase sequence. Thismutation surprisingly does not significantly alter the uricase activityof the protein.

In an embodiment of the invention, amino acids at or around any of aminoacids 7, 46, and 291, and 301 are mutated. In a preferred embodiment ofthe invention, amino acids 7, 46, and 291, and 301, themselves, aremutated.

In particular embodiments, the protein is encoded by a nucleic acid thatencodes an N-terminal rnethionine. Preferably, the N-terminal methionineis followed by a codon that allows for removal of this N-terminalmethionine by bacterial methionine aminopeptidase (MAP). (Ben-Bassat andBauer (1987) Nature 326:315, incorporated herein by reference in itsentirety). Amino acids allowing the most complete removal of theN-terminal methionine are alanine, glycine, proline, serine, andthreonine.

In an embodiment of the invention, the amino acids at or aroundpositions 7 and/or 46 are substituted by threonine, Surprisingly, theenzymatic activity of truncated uricases prepared with these mutationsis similar to that of the non-truncated enzyme. In a further embodimentof the invention, the amino acid mutations comprise threonine,threonine, lysine, and serine, at positions 7, 46, 291, and 301,respectively.

The truncated mammalian uricases disclosed herein may further comprise amethionine at the amino terminus. The penultimate amino acid may onethat allows removal of the N-terminal methionine by bacterial methionineaminopeptidase (MAP). Amino acids allowing the most complete removal ofthe N-terminal methionine are alanine, glycine, proline, serine, andthreonine. In a particular embodiment, the uricase comprises two aminoterminal amino acids, wherein the two amino terminal amino acids are amethionine followed by an amino acid selected from the group consistingof alanine, glycine, proline, serine, and threonine.

In another embodiment of the invention, the substituted amino acids havebeen replaced by threonine.

In an embodiment of the invention, the uricase is a mammalian uricase.

In an embodiment of the invention, the mammalian uricase comprises thesequence of porcine, bovine, ovine or baboon liver uricase.

In an embodiment of the invention, the uricase is a chimeric uricase oftwo or more mammalian uricases.

In an embodiment of the invention, the mammalian uricases are selectedfrom porcine, bovine, ovine, or baboon liver uricase.

In an embodimentof the. invention, the uricase comprises the sequence ofSEQ ID NO. 8.

In another embodiment of the invention, the uricase comprises thesequence of SEQ ID NO. 13.

The subject invention provides uricase encoding nucleic acids comprisingthe sequence of SEQ ID NO. 10.

In an embodiment of the invention, the uricase comprises fungal ormicrobial uricase.

In an embodiment of the invention, the fungal or microbial uricase isAspergillus flavus, Arthrobacter globiformis or Candida utilis uricase.

In an embodiment of the invention, the uricase comprises an invertebrateuricase.

In an embodiment of the invention, the invertebrate uricase Drosophilamelanogaster or Drosophila pseudoobscura uricase.

In an embodiment of the invention, the uricase comprises plant uricase.

In an embodiment of the invention, the planturicase is Glycine maxuricase of root nodules.

The subject invention provides a nucleic acid sequence encoding theuricase.

The subject invention provides a vector comprising the nucleic acidsequence.

In a particular embodiment, the uricase is isolated. In a particularembodiment, the uricase is purified. In particular embodiments, theuricase is isolated and purified.

The subject invention provides a host cell comprising a vector.

The subject invention provides a method for producing the nucleic acidsequence, comprising modification by PCR (polymerase chain reaction)techniques of a nucleic acid sequence encoding a nontruncated uricase.One skilled in the art knows that a desired nucleic acid sequence isprepared by PCR via synthetic oligonucleotide primers, which arecomplementary to regions of the target DNA (one for each strand) to beamplified. The primers are added to the target DNA (that need not bepure), in the presence of excess deoxynucleotides and Taq polymerase, aheat stable DNA polymerase. In a series (typically 30) of temperaturecycles, the target DNA is repeatedly denatured (around 90° C.), annealedto the primers (typically at 50-60° C.) and a daughter strand extendedfrom the primers (72° C.). As the daughter strands themselves act astemplates for subsequent cycles, DNA fragments matching both primers areamplified exponentially, rather than linearly.

The subject invention provides a method for producing a mutantrecombinant uricase comprising transfecting a host cell with the vector,wherein the host cell expresses the uricase, isolating the mutantrecombinant uricase from the host cell, isolating the purified mutantrecombinant uricase using, for example, chromatographic techniques, andpurifying the mutant recombinant uricase. For example, the uricase canbe made according to the methods described in International PatentPublication No. WO 00/08196, incorporated herein by reference in itsentirety.

The uricase may be isolated and/or purified by any method known to thoseof skill in the art. Expressed polypeptides of this invention aregenerally isolated in substantially pure form. Preferably, thepolypeptides are isolated to a purity of at least 80% by weight, morepreferably to a purity of at least 95% by weight, and most preferably toa purity of at least 99% by weight. In general, such purification may beachieved using, for example, the standard techniques of ammonium sulfatefractionation, SDS-PAGE electrophoresis, and affinity chromatography.The uricase is preferably isolated using a cationic surfactant, forexample, cetyl pyridinium chloride (CPC) according to the methoddescribed in copendingl U.S. patent application filed on Apr. 11, 2005having application No. 60/670,520 and attorney docket number103864.146644, entitled Purification Of Proteins With CationicSurfactant, incorporated herein by reference in its entirety.

In a preferred embodiment, the host cell is treated so as to cause theexpression of the mutant recombinant uricase. One skilled in the artknows that transfection of cells with a vector is usually accomplishedusing DNA precipitated with calcium ions, though a variety of othermethods can be used (e.g. electroporation).

In an embodiment of the invention, the vector is under the control of anosmotic pressure sensitive promoter. A promoter is a region of DNA towhich RNA polymerase binds before initiating the transcription of DNAinto RNA. An osmotic pressure sensitive promoter initiates transcriptionas a result of increased osmotic pressure as sensed by the cell.

In an embodiment of the invention, the promoter is a modified osmBpromoter.

In particular embodiments, the uricase of the invention is a uricaseconjugated with a polymer.

In an embodiment of the invention, a pharmaceutical compositioncomprising the uricase is provided. In one embodiment, the compositionis a solution of uricase. In a preferred embodiment, the solution issterile and suitable for infection. In one embodiment, such compositioncomprises uricase as a solution in phosphate buffered saline. In oneembodiment, the composition is provided in a vial, optionally having arubber injection stopper. In particular embodiments, the compositioncomprises uricase in solution at a concentration of from 2 to 16milligrams of uricase per milliliter of solution, from 4 to 12milligrams per milliliter or from 6 to 10 milligrams per milliliter. Ina preferred embodiment, the composition comprises uricase at aconcentration of 8 milligrams per milliliter. Preferably, the mass ofuricase is measured with respect to the protein mass.

Effective administration regimens of the compositions of the inventionmay be determined by one of skill in the art. Suitable indicators forassessing effectiveness of a given regimen are known to those of skillin the art. Examples of such indicators include normalization orlowering of plasma uric acid levels (PUA) and lowering or maintenance ofPUS to 6.8 mg/dL or less, preferably 6 mg/dL or less. In a preferredembodiment, the subject being treated with the composition of theinvention has a PUA of 6 mg/ml or less for at least 70%, at least 80%,or at least 90% of the total treatment period. For example, for a 24week treatment period, the subject preferably has a PUA of 6 mg/ml orless for at least 80% of the 24 week treatment period, i.e., for atleast a time equal to the amount of time in 134.4 days (24 weeks×7days/week×0.8=134.4 days)

In particular embodiments, 0.5 to 24 mg of uricase in solution isadministered once every 2 to 4 weeks. The uricase may he administered inany appropriate way known to one of skill in the art, for example,intravenously, intramuscularly or subcutaneously. Preferably, when theadministration is intravenous, 0.5 mg to 12 mg of uricase isadministered. Preferably, when the administration is subcutaneous, 4 to24 mg of uricase is administered. In a preferred embodiment, the uricaseis administered by intravenous infusion over a 30 to 240 minute period.In one embodiment, 8 mg of uricase is administered once every two weeks.In particular embodiments, the infusion can be performed using 100 to500 mL of saline solution. In a preferred embodiment, 8 mg of uricase insolution is administered over a 120 minute period once every 2 weeks oronce every 4 weeks; preferably the uricase is dissolved in 250 mL ofsaline solution for infusion. In particular embodiments, the uricaseadministrations take place over a treatment period of 3 months, 6months, 8 months or 12 months. In other embodiments, the treatmentperiod is 12 weeks, 24 weeks, 36 weeks or 48 weeks. In a particularembodiment, the treatment period is for an extended period of time,e.g., 2 years or longer, for up to the life of subject being treated. Inaddition, multiple treatment periods may be utilized interspersed withtimes of no treatment, e.g., 6 months of treatment followed by 3 monthswithout treatment, followed by 6 additional months of treatment, etc.

In certain embodiments, anti-inflammatory compounds may beprophylactically administered to eliminate or reduce the occurrence ofinfusion reactions due to the administration of uricase. In oneembodiment, at least one corticosteroid, at least one antihistamine, atleast one NSAID, or combinations thereof are so administered, Usefulcorticosteroids include betamethasone, budesonide, cortisone,dexamethasone, hydrocortisone, methylprednisolone, prednisolone,prednisone and triamcinolone. Useful NSAIDs include ibuprofen,indomethacin, naproxen, aspirin, acetominophen, celecoxib andvaldecoxib. Useful antihistamines include azatadine, brompheniramine,cetirizine, chlorpheniramine, clemastine, cyproheptadine, desloratadine,dexchlorpheniramine, dimenhydrinate, diphenhydramine, doxylamine,fexofenadine, hydroxyzine, loratadine and phenindamine.

In a preferred embodiment, the antihistamine is fexofenadine, the NSAIDis acetaminophen and the corticosteroid is hydrocortisone and/orprednisone. Preferably, a combination of all three (not necessarilyconcomitantly) are administered prior to infusion of the uricasesolution. In a preferred embodiment, the NSAID and antihistamine areadministered orally 1 to 4 hours prior to uricase infusion. A suitabledose of fexofenadine includes about 30 to about 180 mg, about 40 toabout 150 mg, about 50 to about 120 mg, about 60 to about 90 mg, about60 mg, preferably 60 mg. A suitable dose of acetaminophen includes about500 to about 1500 mg, about 700 to about 1200 mg, about 800 to about1100 mg, about 1000 mg, preferably 1000 mg. A suitable dose ofhydrocortisone includes about 100 to about 500 mg, about 150 to about300 mg, about 200 mg, preferably 200 mg. In one embodiment, theantihistamine is not diphenhydramine. In another embodiment, the NSAIDis not acetaminophen, In a preferred embodiment, 60 mg fexofenadine isadministered orally the night before uricase infusion; 60 mgfexofenadine and 1000 mg of acetaminophen are administered orally thenext morning, and finally, 200 mg hydrocortisone is administered justprior to the infusion of the uricase solution. In one embodiment,prednisone is administered the day, preferably in the evening, prior touricase administration. An appropriate dosage of prednisone includes 5to 50 mg, preferably 20 mg. In certain embodiments, these prophylactictreatments to eliminate or reduce the occurrence of infusion reactionsare utilized for subjects receiving or about to receive uricase,including PEGylated uricase and non-PEGylated uricase. In particularembodiments, these prophylactic treatments are utilized for subjectsreceiving or about to receive therapeutic peptides other than uricase,wherein the other therapeutic peptides are PEGylated or non-PEGylated.

In an embodiment of the invention, the pharmaceutical compositioncomprises a uricase that has been modified by conjugation with apolymer, and the modified uricase retains uricolytic activity. In aparticular embodiment, polymer-uricase conjugates are prepared asdescribed in International Patent Publication No. WO 01/59078 and U.S.application Ser. No. 09/501,730, incorporated herein by reference intheir entireties.

In an embodiment of the invention, the polymer is selected from thegroup comprising polyethylene glycol, dextran, polypropylene glycol,hydroxypropylmethyl cellulose, carboxymethylcellulose, polyvinylpyrrolidone, and polyvinyl alcohol.

In an embodiment of the invention, the composition comprises 2-12polymer molecules on each uricase subunit, preferably 3 to 10 polymermolecules per uricase subunit.

In an embodiment of the invention, each polymer molecule has a molecularweight between about 1 kD and about 100 kD.

In another embodiment of the invention, each polymer molecule has amolecular weight between about 1 kD and about 50 kD. In a preferredembodiment of the invention, each polymer molecule has a molecularweight of between about 5 kD and about 20 kD, about 8 kD and about 15kD, about 10 kD and 12 kD, preferably about 10 kD. In a preferredembodiment, each polymer molecule has a molecular weight of about 5 kDor about 20 kD. In an especially preferred embodiment of the invention,each polymer molecule has a molecular weight of 10 kD. Mixtures ofdifferent weight molecules are also contemplated. In an embodiment ofthe invention, the composition is suitable for repeated administrationof the composition.

In a particular embodiment, conjugation of the uricase to the polymercomprises linkages selected from the group consisting of urethanelinkages, secondary amine linkages, and amide linkages.

The subject invention provides a cell with the capacity for producing auricase having an amino acid sequence of recombinant uricase, whereinthe uricase has been truncated by 1-20 amino acids, and has mutatedamino acids and uricolytic activity.

The subject invention provides a means for metabolizing uric acid usingthe uricase.

The subject invention provides a use of a composition of uricase forreducing uric acid levels in a biological fluid.

In an embodiment of the invention, the composition of uricase is usedfor reducing uric acid in a biological fluid comprising blood.

Also provided are novel nucleic acid molecules encoding uricasepolypeptides. The manipulations which result in their production arewell known to the one of skill in the art. For example, uricase nucleicacid sequences can be modified by any of numerous strategies known inthe art (Maniatis, T., 1990, Molecular Cloning, A Laboratory Manual, 2ded., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). Thesequence can be cleaved at appropriate sites with restrictionendonuclease(s), followed by further enzymatic modification if desired,isolated, and ligated in vitro. In the production of the gene encoding auricase, care should be taken to ensure that the modified gene remainswithin the appropriate translational reading frame, uninterrupted bytranslational stop signals. Additionally, the uricase-encoding nucleicacid sequence can be mutated in vitro or in vivo, to create and/ordestroy translation, initiation, and/or termination sequences, or tocreate variations in coding regions and/or form new restrictionendonuclease sites or destroy preexisting ones, to facilitate further invitro modification, Any technique for mutagenesis known in the art canbe used, including but not limited to, in vitro site-directedmutagenesis (Hutchinson, C., et al., 1978, J. Biol. Chem 253:6551), useof TAB® linkers (Pharmacia) (as described in U.S. Pat. No.4,719,179),etc.

The nucleotide sequence coding for a uricase protein can be insertedinto an appropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedprotein-coding sequence, A variety of host-vector systems may beutilized to express the protein-coding sequence. These include but arenot limited to mammalian cell systems infected with virus (e.g.,vaccinia virus, adenovirus, etc.); insect cell systems infected withvirus (e.g., baculovirus); microorganisms such as yeast containing yeastvectors, or bacteria transformed with bacteriophage DNA, plasmid DNA, orcosmid DNA. The expression elements of these vectors vary in theirstrengths and specificities.

Depending on the host-vector system utilized, any one of a number ofsuitable transcription and translation elements may be used.

Any of the methods known for the insertion of DNA fragments into avector may be used to construct expression vectors containing a chimericgene consisting of appropriate transcriptional/translational controlsignals and the protein coding sequences. These methods may include invitro recombinant DNA and synthetic techniques and in vivorecombinations (genetic recombination). Expression of nucleic acidsequence encoding uricase protein may be regulated by a second nucleicacid sequence so that uricase protein is expressed in a host transformedwith the recombinant DNA molecule. For example, expression of uricasemay be controlled by any promoter/enhancer element known in the art.Promoters which may be used to control uricase expression include, butare not limited to, the SV40 early promoter region (Bemoist and Chambon,1981, Nature 290:304-310), the promoter contained in the 3′ longterminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981,Proc. Natl. Acad. Sci. U.S.A. 78:144-1445), the regulatory sequences ofthe metal lothionine gene (Brinster et al., 1982, Nature 296:39-42);prokaryotic expression vectors such as the β-lactamase promoter(Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A.75:3727-3731), the tac promoter (DeBoer, et al., 1983, Proc. Natl. Acad.Sci. U.S.A. 80:21-25), and the osmB promoter. In particular embodiments,the nucleic acid comprises a nucleic acid sequence encoding the uricaseoperatively linked to a heterologous promoter.

Once a particular recombinant DNA molecule comprising a nucleic acidsequence encoding is prepared and isolated, several methods known in theart may be used to propagate it. Once a suitable host system and growthconditions are established, recombinant expression vectors can bepropagated and prepared in quantity. As previously explained, theexpression vectors which can be used include, but are not limited to,the following vectors or their derivatives: human or animal viruses suchas vaccinia virus or adenovirus; insect viruses such as baculovirus;yeast vectors; bacteriophage vectors (e.g., lambda), and plasmid andcosmid DNA vectors, to name but a few.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Expression from certainpromoters can be elevated in the presence of certain inducers; thus,expression of the genetically engineered uricase protein may becontrolled. Furthermore, different host cells have characteristic andspecific mechanisms for the translational and post-translationalprocessing and modification (e.g., glycosylation, cleavage) of proteins.Appropriate cell lines or host systems can be chosen to ensure thedesired modification and processing of the foreign protein expressed.Different vector/host expression systems may effect processing reactionssuch as proteolytic cleavages to different extents.

In particular embodiments of the invention, expression of uricase in E.coli is preferably performed using vectors which comprise the osmBpromoter.

EXAMPLES Example 1 Construction of Gene and Expression Plasmid forUricase Expression

Recombinant porcine uricase (urate oxidase), Pig-KS-ΔN (amino terminustruncated pig uricase protein replacing amino acids 291 and 301 withlysine and serine, respectively) was expressed in E. coli K-12 strain W3110 F−. A series of plasmids was constructed culminating inpOUR-P-ΔN-ks-1, which upon transformation of the E. coli host cells wascapable of directing efficient expression of uricase.

Isolation and Subcloning of Uricase cDNA from Pig and Baboon Liver

Uricase cDNAs were prepared from pig and baboon livers by isolation andsubcloning of the relevant RNA. Total cellular RNA was extracted frompig and baboon livers (Erlich, H. A. (1989). PCR Technology; Principlesand Application for DNA Amplification; Sambrook, J., et al. (1989).Molecular Cloning: A Laboratory Manual, 2nd edition; Ausubel, F. M. etal. (1998). Current protocols in molecular Biology), thenreverse-transcribed using the First-Strand cDNA Synthesis Kit (PharmaciaBiotech). PCR amplification was performed using Tag DNA polymerase(Gibco BRL, Life Technologies).

The synthetic oligonucleotide primers used for PCR amplification of pigand baboon urate oxidases incase are shown in Table 1.

TABLE 1 Primers For PCR Amplification Of Uricase cDNA Pig liver uricase:sense 5′ gcgcgaattccATGGCTCATTACCGTAATGACTACA 3′ (SEQ ID NO. 1)anti-sense 5′ gcgctctagaagcttccatggTCACAGCCTTGAAGTCAGC 3′ (SEQ ID NO. 2) Baboon (D3H) liver uricase: sense5′ gcgcgaattccATGGCCCACTACCATAACAACTAT 3′ (SEQ ID NO. 3) anti-sense5′ gcgcccatggtctagaTCACAGTCTTGAAGACAACT TCCT 3′ (SEQ ID NO. 4)

Restriction enzyme sequences, introduced at the ends of the primers andshown in lowercase in Table 1, were sense EcoRI and NcoI (pig andbaboon) and anti-sense NcoI, HindIII and XbaI (pig), XbaI and NcoI(baboon). In the baboon sense primer, the third codon GAC (asparticacid) present in baboon uricase was replaced with CAC (histidine), thecodon that is present at this position in the coding sequence of thehuman urate oxidase pseudogene. The recombinant baboon uricase constructgenerated using these primers is named D3H Baboon Uricase.

The pig uricase PCR product was digested with EcoRI and HindIII andcloned into pUC18 to create pUC18-Pig Uricase. The D3H Baboon UricasePCR product was cloned directly into PCR®II vector (TA Cloning VectorpCR™II), using TA Cloning® biochemical laboratory kits for cloning ofamplified nucleic acids (Invitrogen, Carlsbad, Calif.), creatingPCR®II-D3H Baboon Uricase.

Ligated cDNAs were used to transform E. coli strain XL1-Blue(Stratagene, La Jolla, Calif.). Plasmid DNA containing cloned uricasecDNA was prepared, and clones which possess the published uricase DNAcoding sequences (except for the D3H substitution in baboon uricase,shown in Table 1) were selected and isolated. In the PCR®II-D3H BaboonUricase clone chosen, the PCR®II sequences were next to the uricase stopcodon, resulting from deletion of sequences introduced by PCR. As aconsequence, the XbaI and NcoI restriction sites from the 3′untranslated region were eliminated, thus allowing directional cloningusing INcol at the 5′ end of the PCR product and BamHI which is derivedfrom the PCR®II vector.

Subcloning of Uricase cDNA into pET Expression Vectors Baboon UricaseSubcloning

The D3H baboon cDNA containing full length uricase coding sequence wasintroduced into pET-3d expression vector (Novagen, Madison, Wis.). ThePCR®II-D3H Baboon Uricase was digested with NcoI and BamHI, and the 960bp fragment was isolated. The expression plasmid pET-3d was digestedwith NcoI and BamHI, and the 4600 bp fragment was isolated. The twofragments were ligated to create pET-3d-D3H-Baboon.

Pig-Baboon Chimera Uricase Subcloning

Pig-baboon chimera (PBC) uricase was constructed in order to gain higherexpression, stability, and activity of the recombinant gene. PBC wasconstructed by isolating the 4936 bp NcoI-ApaI fragment frompET-3d-D3H-Baboon clone and ligating the isolated fragment with the 624by NcoI-ApaI fragment isolated from pUC18-Pig Uricase, resulting in theformation of pET-3d-PBC. The PBC uricase cDNA consists of the piguricase codons 1-225 joined in-frame to codons 226-304 of baboonuricase.

Pig-KS Uricase Subcloning

Pig-KS uricase was constructed in order to add one lysine residue, whichmay provide an additional PEGylation site. KS refers to the amino acidinsert of lysine into pig uricase, at position 291, in place of arginine(R291K). In addition, the threonine at position 301 was replaced withserine (T301 S). The PigKS uricase plasmid was constructed by isolatingthe 4696 bp NcoI-NdeI fragment of pET-3d-D3H-Baboon, and then it wasligated with the 864 by NcoI-NdeI fragment isolated from pUC18-PigUricase, resulting in the formation of pET-3d-PigKS. The resulting PigKSuricase sequence consists of the pig uricase codons 1-288 joinedin-frame to codons 289-304 of baboon uricase.

Subcloning of Uricase Sequence Under the Regulation of the osmB Promoter

The uricase gene was subcloned into an expression vector containing theosmB promoter (following the teaching of U.S. Pat. No. 5,795,776,incorporated herein by reference in its entirety). This vector enabledinduction of protein expression in response to high osmotic pressure orculture aging. The expression plasmid pMFOA-18 contained the osmBpromoter, a ribosotnal binding site sequence (rbs) and a transcriptionterminator sequence (ter). It confers ampicillin resistance (AmpR) andexpresses the recombinant human acetylcholine esterase (AChE).

Subcloning of D3H-Baboon Uricase

The plasmid pMFOA-18 was digested with NcoI and BamHI, and the largefragment was isolated. The construct pET-3d-D3H-Baboon was digested withNcoI and BamHI and the 960 bp fragment, which included the D3H BaboonUricase gene is isolated. These two fragments were ligated to createpMFOU18.

The expression plasmid pMFXT133 contained the osmB promoter, a rbs (E.coli deo operon), ter (E. coli TrypA), the recombinant factor Xainhibitor polypeptide (FxaI, and it 2 5 conferred the tetracyclineresistance gene (TetR). The baboon uricase gene was inserted into thisplasmid in order to exchange the antibiotic resistance genes. Theplasmid pMFOU18 was digested with NcoI, filled-in, then it was digestedwith XhoI, and a 1030 by fragment was isolated. The plasmid pMFXT133 wasdigested with NdeI, filled-in, then it was digested with XhoI, and thelarge fragment was isolated. The two fragments were ligated to createthe baboon uricase expression vector, pURBA16.

Subcloning of the Pig Baboon Chimera Uricase

The plasmid pURBA16 was digested with ApaI and AlwNI, and the 2320 byfragment was isolated. The plasmid pMFXT133 was digested with NdeI,filled-in, then it was digested with AlwNI, and the 620 bp fragment wasisolated. The construct pET-3d-PBC was digested with XbaI, filled-in,then it was digested with ApaI, and the 710 by fragment was isolated.The three fragments were ligated to create pUR-PB, a plasmid thatexpressed PBC uricase under the control of osmB promoter and rbs as wellas the T7 rbs, which was derived from the pET-3d vector.

The T7 rbs was excised in an additional step. pUR-PB was digested withNcoI, fitted-in, then digested with AlwNI, and the 3000 by fragment wasisolated. The plasmid pMFXT133 was digested with NdeI, filled in andthen digested with AIwNI, and the 620 bp fragment was isolated. The twofragments were ligated to form pDUR-PB, which expresses PBC under thecontrol of the osmB promoter.

Construction of pOUR-PB-ΔNC

Several changes were introduced into the uricase cDNA, which resulted ina substantial increase in the recombinant enzyme stability. PlasmidpOUR-PBC-ΔNC was constructed, in which the N-terminal six-residuematuration peptide and the tri-peptide at the C-terminus, which functionin vivo as peroxysomal targeting signal, were both removed. This wascarried out by utilizing PBC sequence in plasmic pDUR-PB and thespecific oligonucleotide primers listed in Table 2, using PCRamplification.

TABLE 2 Primers for PCR Amplification of PBC-ΔNC UricasePBC-ΔNC Uricase: Sense 5′ gcgcatATGACTTACAAAAAGAATGATGAGGTAGAG3′ (SEQ ID NO. 5) Anti-sense 5′ ccgtctagaTTAAGACAACTTCCTCTTGACTGTACCAGTAATTTTTCCGTATGG 3′ (SEQ ID NO. 6)

The restriction enzyme sequences introduced at the ends of the primersshown in bold and the non-coding regions are shown in lowercase in Table2. NdeI is sense and XbaI is anti-sense. The anti-sense primer was alsoused to eliminate an internal NdeI restriction site by introducing apoint mutation (underlined) which did not affect the amino acidsequence, and thus, facilitated subcloning by using NdeI.

The 900 base-pair fragment generated by PCR amplification of pDUR-PB wascleaved with NdeI and XbaI and isolated. The obtained fragment was theninserted into a deo expression plasmic pDBAST-RAT-N, which harbors thedeo-P1P2 promoter and rbs derived from E. coli and constitutivelyexpresses human recombinant insulin precursor. The plasmid was digestedwith NdeI and XbaI and the 4035 by fragment was isolated and ligated tothe PBC-uricase PCR product. The resulting construct, pDUR-PB-ΔNC, wasused to transform E. coli K-12 Sφ733 (F-cytR strA) that expressed a highlevel of active truncated uricase.

The doubly truncated PBC-ΔNC sequence was also expressed under thecontrol of osmB promoter. The plasmid pDUR-PB-ΔNC was digested withAlwNI-NdeI, and the 3459 bp fragment was isolated. The plasmidpMFX.T133, described above, was digested with NdeI-AlwNI, and the 660 byfragment was isolated. The fragments were then ligated to createpOUR-PB-ΔNC, which was introduced into E. coli K-12 strain W3110 F⁻ andexpressed high level of active truncated uricase.

Construction of the Uricase Expression Plasmid pOUR-P-ΔN-ks-1

This plasmid was constructed in order to improve the activity andstability of the recombinant enzyme. Pig-KS-ΔN uricase was truncated atthe N-terminus only (ΔN), where the six-residue N-terminal maturationpeptide was removed, and contained the mutations S46T, R291K and T301S.At position 46, there was a threonine residue instead of serine due to aconservative mutation that occurred during PCR amplification andcloning, At position 291, lysine replaced arginine, and at position 301,serine was inserted instead of threonine. Both were derived from thebaboon uricase sequence. The modifications of R291K and T3015 aredesignated KS, and discussed above. The extra lysine residue provided anadditional potential PEGylation site.

To construct pOUR-P-ΔN-ks-1 (FIG. 1), the plasmid pOUR-PB-ΔNC wasdigested with ApaI-XbaI, and the 3873 bp fragment was isolated. Theplasmid pET-3d-PKS (construction shown in FIG. 4) was digested withApaI-SpeI, and the 270 bp fragment was isolated. SpeI cleavage left a 5′CTAG extension that was efficiently ligated to DNA fragments generatedby XbaI. The two fragments were ligated to create pOUR-P-ΔN-ks-1. Afterligation, the SpeI and XbaI recognition sites were lost (their site isshown in parenthesis in FIG. 9). The construct pOUR-P-ΔN-ks-1 wasintroduced into E. coli K-12 strain W3110 F⁻, prototrophic, ATCC #27325.The resulting Pig-KS-ΔN uricase, expressed under the control of osmBpromoter, yielded high levels of recombinant enzyme having superioractivity and stability.

FIG. 1 illustrates the structure of plasmid pOUR-P-ΔN-ks-1. Numbers nextto restriction sites indicate nucleotide position, relative to HaeIIsite, designated as 1; restriction sites that were lost during cloningare marked in parenthesis. Plasmid pOUR-P-ΔN-ks-1, encoding Pig-KS-ΔNuricase is 4143 base pairs (bp) long and comprised the followingelements:

-   -   1. A DNA fragment, 113 bp long, spanning from nucleotide number        1 to NdeI site (at position 113), which includes the osmB        promoter and ribosome binding site (rbs).    -   2. A DNA fragment, 932 by long, spanning from NdeI (at        position 113) to SepI/XbaI junction (at position 1045), which        includes: 900 bp of Pig-KS-ΔN (nucleic acid sequence of amino        terminus truncated pig uricase protein in which amino acids 291        and 301 with lysine and serine, respectively, are replaced)        coding region and 32 bp flanking sequence derived from pCR™II,        from the TA cloning site upstream to the SpeI/XbaI restriction        site.    -   3. A 25 bp multiple cloning sites sequence (MCS) from SpeI/XbaI        junction (at position 1045) to HinclER (at position 1070).    -   4. A synthetic 40 bp oligonucleotide containing the TrpA        transcription terminator (ter) with HindIII (at position 1070)        and AatII (at position 1110) ends.    -   5. A DNA fragment, 1519 by long, spanning from AatII (at        position 1110) to MscI/ScaI (at position 2629) sites on pBR322        that includes the tetracycline resistance gene (TetR).    -   6. A DNA fragment, 1514 by long, spanning from ScaI (at        position 2629) to HaeII (at position 4143) sites on pBR322 that        includes the origin of DNA replication.

FIG. 2 shows the DNA and the deduced amino acid sequences of Pig-KS-ΔNuricase. In this figure, the amino acid numbering is according to thecomplete pig uricase sequence. Following the initiator methionineresidue, a threonine was inserted in place of the aspartic acid of thepig uricase sequence. This threonine residue enabled the removal ofmethionine by bacterial aminopeptidase. The gap in the amino acidsequence illustrates the deleted N-terminal maturation peptide. Therestriction sites that were used for the various steps of subcloning ofthe different uricase sequences (ApaI, NdeI, BamHI, RcoRI and SpeI) areindicated. The 3° untranslated sequence, shown in lowercase letters, wasderived from PCR®II sequence. The translation stop codon is indicated byan asterisk.

FIG. 3 shows alignment of the amino acid sequences of the variousrecombinant uricase sequences. The upper line represents the piguricase, which included the full amino acid sequence. The second line isthe sequence of the doubly truncated pig-baboon chimera uricase(PBC-ΔNC). The third line shows the sequence of Pig-KS-ΔN uricase, thatis only truncated at the N-terminus and contained the mutations S46T andthe amino acid changes R291K and T301 S, both reflecting the baboonorigin of the carboxy terminus of the uricase coding sequence. Theasterisks indicate the positions in which there are differences in aminoacids in the Pig-KS-ΔN as compared to the published pig uricasesequence; the circles indicate positions in which there are differencesin amino acids in Pig-KS-ΔN compared to PBC-ΔN, the pig-baboon chimera;and dashed lines indicate deletion of amino acids.

cDNA for native baboon, pig, and rabbit uricase with the Y97H mutation,and the pig/baboon chimera (PBC) were constructed for cloning into E.coli. Clones expressing high levels of the uricase variants wereconstructed and selected such that all are W3110 F⁻ E. coli, andexpression is regulated by osmB. Plasmid DNAs were isolated, verified byDNA sequencing and restriction enzyme analysis, and cells were cultured.

Construction of the truncated uricases, including pig-ΔN and Pig-KS-ΔNwas done by cross-ligation between PBC-ΔNC and Pig-KS, followingcleavage with restriction endonucleases ApaI and XbaI, and ApaI plusSpeI, respectively. It is reasonable that these truncated mutants wouldretain activity, since the N-terminal six residues, the “maturationpeptide” (1-2), and the C-terminal tri-peptide, “peroxisomal targetingsignal” (3-5), do not have functions which significantly affectenzymatic activity, and it is possible that these sequences may beimmunogenic. Clones expressing very high levels of the uricase variantswere selected.

Example 2 Transformation of the Expression Plasmid into a Bacterial HostCell

The expression plasmid, pOUR-P-ΔN-ks-1, was introduced into E. coli K-12strain W3110 F⁻ Bacterial cells were prepared for transformationinvolved growth to mid log phase in Luria broth (LB), then cells wereharvested by centrifugation, washed in cold water, and suspended in 10%glycerol, in water, at a concentration of about 3×10¹⁰ cells per ml. Thecells were stored in aliquots, at −70° C. Plasmid DNA was precipitatedin ethanol and dissolved in water.

Bacterial cells and plasmid DNA were mixed, and transformation was doneby the high voltage electroporation method using Gene Pulser II fromBIO-RAD (Trevors et al (1992). Electrotransformation of bacteria byplasmid DNA, in Guide to Electroporation and Electrofusion (D. C. Chang,B. M. Chassy, J. A. Saunders and A. E. Sowers, eds.), pp. 265-290,Academic Press Inc., San Diego, Hanahan et al (1991) Meth. Enzymol.,204, 63-113), Transformed cells were suspended in SOC medium (2%tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mMMgSO₄, 20 mM glucose), incubated, at 37° C., for 1 hour and selected fortetracycline resistance. A high expresser clone was selected.

Example 3 Recombinant Uricase Preparation

Bacteria such as those transformed (see above) were cultured in mediumcontaining glucose; pH was maintained at 7.2±0.2, at approximately 37°C.

Towards the last 5-6 hours of cultivation, the medium was supplementedwith KCl to a final concentration of 0.3M. Cultivation was continued toallow uricase accumulation.

Recombinant uricase accumulated within bacterial cells as an insolubleprecipitate similar to inclusion bodies (IBs). The cell suspension waswashed by centrifugation and suspended in 50 mM Tris buffer, pH 8.0 and10 mM EDTA and brought to a final volume of approximately 40 times thedry cell weight.

Recombinant uricase-containing IBs, were isolated by centrifugationfollowing disruption of bacterial cells using lysozyme and highpressure. Treatment with lysozyme (2000-3000 units/ml) was done for16-20 hours at pH 8.0 and 7±3° C., while mixing. The pellet was washedwith water and stored at −20° C. until use,

The enriched IBs were further processed after suspending in 50 mM NaHCO₃buffer, pH 10.3±0.1. The suspension was incubated overnight, at roomtemperature, to allow solubilization of the IB-derived uricase, andsubsequently clarified by centrifugation.

Uricase was further purified by several chromatography steps. Initially,chromatography was done on a Q-Sepharose FF column. The loaded columnwas washed with bicarbonate buffer containing 150 mM NaCl, and uricasewas eluted with bicarbonate buffer, containing 250 mM NaCl. Then,Xanthine-agarose resin (Sigma) was used to remove minor impurities fromthe uricase preparation. The Q-Sepharose FE eluate was diluted with 50mM glycine buffer, pH 10.3±0.1, to a protein concentration ofapproximately 0.25 mg/ml and loaded. The column was washed withbicarbonate buffer, pH 10.3±0.1, containing 100 mM NaCl, and uricase waseluted with the same buffer supplemented with 60 μM xanthine. At thisstage, the uricase was repurified by Q-Sepharose chromatography toremove aggregated forms.

The purity of each uricase preparation is greater than 95%, asdetermined by size exclusion chromatography. Less than 0.5% aggregatedforms are detected in each preparation using a Superdex 200 column.

Table 3 summarizes purification of Pig-KSΔN uricase from IBs derivedfrom 25 L fermentation broth.

TABLE 3 Purification Of Pig-KSΔN Uricase Protein Activity SpecificPurification step (mg) (U) Activity (U/mg) IB dissolution 12,748 47,2263.7 Clarified solution 11,045 44,858 4.1 Q-Sepharose I-main pool 7,59032,316 4.3 Xanthine Agarose-main pool 4,860 26,361 5.4 Q-SepharoseII-main pool 4,438 22,982 5.2 30 kD UF retentate 4,262 27,556 6.5

Example 4 Characteristics of Recombinant Uricases

SDS-PAGE analysis of the highly purified uricase variants (FIG. 4)revealed a rather distinctive pattern. The samples were stored at 4° C.,in carbonate buffer, pH 10.3, for up to several months. The full-lengthvariants, Pig, Pig-KS, and PBC, show accumulation of two majordegradation products having molecular weights of about 20 and 15 kD.This observation suggests that at least a single nick split the uricasesubunit molecule. A different degradation pattern is detected in theamino terminal shortened clones and also in the rabbit uricase, but at alower proportion. The amino terminus of the rabbit resembles that of theshortened clones. The amino terminal sequences of the uricase fragmentsgenerated during purification and storage were determined.

Peptide Sequencing

N-terminal sequencing of bulk uricase preparations was done using theEdman degradation method. Ten cycles were perfoired. Recombinant Piguricase (full length clone) generated a greater abundance of degradationfragments compared to Pig-KS-ΔN. The deduced sites of cleavage leadingto the degradation fragments are as follows:

-   -   1) Major site at position 168 having the sequence:    -   -QSG ↓FEGFI-    -   2) Minor site at position 142 having the sequence:    -   -IRN ↓GPPVI-

The above sequences do not suggest any known proteolytic cleavage.Nevertheless, cleavage could arise from either proteolysis or somechemical reaction. The amino-truncated uricases are surprisingly morestable than the non-amino truncated uricases. PBCΔNC also had stabilitysimilar to the other ΔN molecules and less than non-amino-truncated PBC.

Potency

Activity of uricase was measured by a UV method. Enzymatic reaction ratewas determined by measuring the decrease in absorbance at 292 nmresulting from the oxidation of uric acid to allantoin. One activityunit is defined as the quantity of uricase required to oxidize one nmoleof uric acid per minute, at 25° C., at the specified conditions. Uricasepotency is expressed in activity units per mg protein (U/mg).

The extinction coefficient of 1 mM uric acid at 292 nm is 12.2 mM⁻¹cm⁻¹. Therefore, oxidation of 1 μmole of uric acid per ml reactionmixture resulted in a decrease in absorbance of 12.2 mA₂₉₂. Theabsorbance change with time (ΔA₂₉₂ per minute) was derived from thelinear portion of the curve.

Protein concentration was determined using a modified Bradford method(Macart and Gerbaut (1982) Clin Chim Acta 122:93-101). The specificactivity (potency) of uricase was calculated by dividing the activityU/ml with protein concentration in mg/ml. The enzymatic activity resultsof the various recombinant uricases are summarized in Table 4. Theresults of commercial preparations are included in this table asreference values. It is apparent from these results that truncation ofuricase proteins has no significant effect on their enzymatic activity.

TABLE 4 Summary of Kinetic Parameters of Recombinant and Native UricasesConcentration⁽¹⁾ Specific of Activity Km ⁽⁴⁾ Kcat⁽⁵⁾ UricasesStock(mg/ml) (U/mg)⁽²⁾ (μM Uric Acid) (l/min) Recombinant Pig 0.49 7.414.39 905 Pig-ΔN 0.54 7.68 4.04 822 Pig-KS 0.31 7.16 5.27 1085 Pig-KS-ΔN1.14 6.20 3.98 972 PBC 0.76 3.86 4.87 662 PBC-ΔNC 0.55 3.85 4.3 580Rabbit 0.44 3.07 4.14 522 Native Pig (Sigma) 2.70 3.26⁽³⁾ 5.85 901 A.flavus (Merck) 1.95 0.97⁽³⁾ 23.54 671 Table 4 Notes: ⁽¹⁾Proteinconcentration was determined by absorbance measured at 278 nm, using anExtinction coefficient of 11.3 for 10 mg/ml uricase solution (Mahler,1963). ⁽²⁾1 unit of uricase activity is defined as the amount of enzymethat oxidizes 1 μmole of uric acid to allantoin per minute, at 25° C.⁽³⁾Specific activity values were derived from the Lineweaver-Burk plots,at a concentration of substrate equivalent to 60 μM. ⁽⁴⁾ ReactionMixtures were composed of various combinations of the following stocksolutions 100 mM sodium borate buffer, pH 9.2 300 μM Uric acid in 50 mMsodium borate buffer, pH 9.2 1 mg/ml BSA in 50 mM sodium borate buffer,pH 9.2 ⁽⁵⁾K_(cat) was calculated by dividing the Vmax (calculated fromthe respective Lineweaver-Burk plots) by the concentration of uricase inreaction mixture (expressed in mol equivalents, based on the tetramericmolecular weights of the uricases).

Example 5 Conjugation of Uricase With m-PEG (PEGylation

Pig-KS-ΔN Uricase was conjugated using m-PEG-NPC(monomethoxy-poly(ethylene glycol)-nitrophenyl carbonate). Conditionsresulting in 2-12 strands of 5, 10, or 20 kD PEG per uricase subunitwere established. m-PEG-NPC was gradually added to the protein solution.After PEG addition was concluded, the uricaselm-PEG-NPC reaction mixturewas then incubated at 2-8° C. for 16-18 hours, until maximal unboundm-PEG strands were conjugated to uricase.

The number of PEG strands per PEG-uricase monomer was determined bySuperose 6 size exclusion chromatography (SEC), using PEG and uricasestandards. The number of bound PEG strands per subunit was determined bythe following equation:

${{PEG}\mspace{14mu} {strands}\text{/}{subunit}} = \frac{3.42 \times {Amount}\mspace{14mu} {of}\mspace{14mu} {PEG}\mspace{14mu} {in}\mspace{14mu} {injected}\mspace{20mu} {sample}\mspace{14mu} ({µg})}{{Amount}\mspace{14mu} {of}\mspace{14mu} {protein}\mspace{14mu} {in}\mspace{14mu} {injected}\mspace{14mu} {sample}\mspace{14mu} ({µg})}$

The concentration of PEG and protein moieties in the PEG-uricase samplewas determined by size exclusion chromatography (SEC) using ultraviolet(UV) and refractive index (RI) detectors arranged in series (asdeveloped by Kunitani, et al., 1991). Three calibration curves aregenerated: a protein curve (absorption measured at 220 nm); a proteincurve (measured by RI); and PEG curve (measured by RI). Then, thePEG-uricase samples were analyzed using the same system. The resultingUV and RI peak area values of the experimental samples were used tocalculate the concentrations of the PEG and protein relative to thecalibration curves. The index of 3.42 is the ratio between the molecularweight of uricase monomer (34,192 Daltons) to that of the 10 kD PEG.

Attached PEG improved the solubility of uricase in solutions havingphysiological values. Table 5 provides an indication of the variabilitybetween batches of PEGylated Pig-KS-ΔN uricase product. In general,there is an inverse relation between the number of PEG strands attachedand retained specific activity (SA) of the enzyme.

TABLE 5 Enzymatic Activity Of PEGylated Pig-KS-ΔN Uricase Conjugates PEGPEG Strands Uricase Conjugate MW per Uricase SA SA Percent Batches (kD)Subunit (U/mg) of Control ΔN-Pig-KS- — — 8.2 100 1-17 # 5 9.7 5.8 70.4LP-17 10 2.3 7.8 94.6 1-15 # 10 5.1 6.4 77.9 13 # 10 6.4 6.3 76.9 14 #10 6.5 6.4 77.5 5-15 # 10 8.8 5.4 65.3 5-17 # 10 11.3 4.5 55.3 4-17 # 1011.8 4.4 53.9 1-18 # 20 11.5 4.5 54.4

Example 6 PEGylation of Uricase with 1000 D and 100,000 D PEG

Pig-KS-ΔN Uricase was conjugated using 1000 D and 100,000 D m-PEG-NPC asdescribed in Example 5. Conditions resulting in 2-11 strands of PEG peruricase subunit were used. After PEG addition was concluded, theuricase/m-PEG-NPC reaction mixture was then incubated at 2-8° C. for16-18 hours, until maximal unbound in-PEG strands were conjugated touricase.

The number of PEG strands per PEG-uricase monomer was determined asdescribed above.

Attached PEG improved the solubility of uricase in solutions havingphysiological pH values.

Example 7 Pharmacokinetics of Pig-KS-ΔN Uricase Conjugated with PEG

Biological experiments were undertaken in order to determine the optimalextent and size of PEGylation needed to provide therapeutic benefit.

Pharmacokinetic studies in rats, using i.v. injections of 0.4 mg (2 U)per kg body weight of unmodified uricase, administered at day 1 and day8, yielded a circulating half life of about 10 minutes. However, studiesof the clearance rate in rats with 2-11×10 kD PEG-Pig-KS-ΔN uricase,after as many as 9 weekly injections, indicated that clearance did notdepend on the number of PEG strands (within this range) and remainedrelatively constant throughout the study period (see Table 6; with ahalf-life of about 30 hours). The week-to-week differences are withinexperimental error. This same pattern is apparent after nine injectionsof the 10×5 kD PEG, and 10×20 kJ) PEG-uricase conjugates. The resultsindicated that regardless of the extent of uricase PEGylation, in thisrange, similar biological effects were observed in the rat model.

TABLE 6 Half Lives of PEGylated Pig-KS-ΔN Uricase Preparations in RatsExtent of Modification (PEG Strands per Uricase Subunit) 5kDPEG 10kD PEG20kD PEG Week 10× 2× 5× 7× 9× 11× 10× 1 25.7 ± 29.4 ± 37.7 ± 37.6 ± 36.9± 31.4 ± 21.6 ± 1.7 3.4 3.1 3.9 4.3 4.3 1.5 (5) (5) (5) (5) (5) (5) (5)2 — — — 26.7 ± 28.4 ± — — 3.0 1.6 (5) (5) 3 27.5 ± 29.0 ± 29.9 ± 32.7 ±26.3 ± 11.8 ± 14.5 ± 3.8 2.6 11.7 11.1 4.7 3.3 2.7 (5) (5) (5) (5) (5)(5) (5) 4 — — 27.1 ± 18.4 ± 19.7 ± — — 5.3 2.2 5.6 — — (5) (4) (4) 528.6 ± 22.5 ± 34.3 ± 37.3 ± 30.4 ± 30.5 ± 19.3 ± 1.7 2.7 3.9 3.0 3.6 1.32.5 (5) (5) (4) (5) (5) (5) (5) 6 — — 35.4 ± 27.1 ± 30.7 ± — — 3.1 3.62.9 (14) (13) (13) 7 16.5 ± 32.5 ± — — — 16.12 ± 25.8 ± 4.9 4.3 2.7 2.5(5) (5) (5) (5) 8 — — — — — — — 9 36.8 ± 28.7 ± 34.0 ± 24.2 ± 31.0 ±29.3 ± 26.7 ± 4.0 2.7 2.4 3.4 2.6 1.4 0.5 (15) (15) (13) (13) (13) (15)(15)

Table 6 notes: Results are indicated in hours±standard error of themean. Numbers in parenthesis indicate the number of animals tested.

Rats received weekly i.v. injections of 0.4 mg per kilogram body weightof Pig-KS-ΔN uricase modified as indicated in the table. Each groupinitially comprised 15 rats, which were alternately bled in subgroups of5. Several rats died during the study due to the anesthesia. Half-liveswere determined by measuring uricase activity (calorimetric assay) inplasma samples collected at, 5 minutes, and 6, 24 and 48 hours postinjection.

Table 5 describes the batches of PEGylated uricase used in the study.

Bioavailability studies with 6×5 kD PEG-Pig-KS-ΔN uricase in rabbitsindicate that, after the first injection, the circulation half-life is98.2±1.8 hours (i.v.), and the bioavailability after i.m. andsubcutaneous (s.c.) injections was 71% and 52%, respectively, However,significant anti-uricase antibody titers were detected, after the secondi.m. and s.c. injections, in all of the rabbits, and clearance wasaccelerated following subsequent injections. Injections of rats with thesame conjugate resulted in a half-life of 26±1.6 hours (i.v.), and thebioavailability after i.m. and s.c. injections was 33% and 22%,respectively.

Studies in rats, with 9×10 kD PEG-Pig-KS-ΔN uricase indicate that thecirculation half-life after the first injection is 42.4 hours (i.v.),and the bioavailability, after i.m. and s.c. injections, was 28.9% and14.5%, respectively (see FIG. 5 and Table 7). After the fourthinjection, the circulation half-life was 32.1±2.4 hours and thebioavailability, after the i.m. and s.c. injections was 26.1% and 14.9%,respectively.

Similar pharmacokinetic studies, in rabbits, with 9×10 kD PEG-Pig-KS-ΔNuricase indicate that no accelerated clearance was observed followinginjection of this conjugate (4 biweekly injections were administered).In these animals, the circulation half-life after the first injectionwas 88.5 hours (i.v.), and the bioavailability, after i.m. and s.c.injections, was 98.3% and 84.4%, respectively (see FIG. 6 and Table 7).After the fourth injection the circulation half-life was 141.1±15.4hours and the bioavailability, after the i.m. and s.c. injections was85% and 83%, respectively.

Similar studies with 9×10 kD PEG-Pig-KS-ΔN were done to assess thebioavailability in beagles males and 2 females in each group). Acirculation half-life of 7±11.7 hours was recorded after the first i.v.injection, and the bioavailability, after the i.m. and s.c. injectionswas 69.5% and 50.4%, respectively (see FIG. 7 and Table 7).

Studies with 9×10 kD PEG-Pig-KS-ΔN preparations were done using pigs.Three animals per group were used for administration via the i.v., s.c.and i.m. routes. A circulation half-life of 178±24 hours was recordedafter the first i.v. injection, and the bioavailability, after the i.m.and s.c. injections was 71.6% and 76.8%, respectively (see FIG. 8 andTable 7).

TABLE 7 Pharmacokinetic Studies with 9 × 10 kD PEG-Pig-KS-ΔN UricaseHalf-life (hours) Bioavailability Injection # i.v. i.m. s.c. Rats 1 42.4± 4.3 28.9% 14.5% 2 24.1 ± 5.0 28.9% 14.5% 4 32.1 ± 2.4 26.1% 14.9%Rabbits 1 88.5 ± 8.9 98.3% 84.4% 2  45.7 ± 40.6  100%  100% 4 141.1 ±15.4   85%   83% Dogs 1  70.0 ± 11.7 69.5% 50.4% Pigs 1 178 ± 24 71.6%76.8%

Absorption, distribution, metabolism, and excretion (ADME) studies weredone after iodination of 9×10 kD PEG-Pig-KS-ΔN uricase by the Bolton &Hunter method with ¹²⁵I. The labeled conjugate was injected into 7groups of 4 rats each (2 males and 2 females). Distribution ofradioactivity was analyzed after 1 hour and every 24 hours for 7 days(except day 5). Each group, in its turn, was sacrificed and thedifferent organs were excised and analyzed. The seventh group was keptin a metabolic cage, from which the urine and feces were collected. Thedistribution of the material throughout the animal's body was evaluatedby measuring the total radioactivity in each organ, and the fraction ofcounts (kidney, liver, lung, and spleen) that were available forprecipitation with TCA (i.e. protein bound, normalized to the organsize). Of the organs that were excised, none had a higher specificradioactivity than the others, thus no significant accumulation was seenfor instance in the liver or kidney, 70% of the radioactivity wasexcreted by day 7.

Example 8 Clinical Trial Results

A randomized, open-label, multicenter, parallel group study wasperformed to assess the urate response, and pharmacokinetic and safetyprofiles of PEG-uricase (Puricase®, Savient Pharmaceuticals) in humanpatients with hyperuricemia and severe gout who were unresponsive to orintolerant of conventional therapy. The mean duration of disease was 14years and 70 percent of the study population had one or more tophi.

In the study, 41 patients (mean age of 58.1 years) were randomized to 12weeks of treatment with intravenous PEG-uricase at one of four doseregimens: 4 mg every two weeks (7 patients); 8 mg every two weeks (8patients); 8 mg every four weeks (13 patients); or 12 mg every fourweeks (13 patients). Plasma uricase activity and urate levels weremeasured at defined intervals. Pharmacokinetic parameters, mean plasmaurate concentration and the percentage of time that plasma urate wasless than or equal to 6 mg/dL were derived from analyses of the uricaseactivities and urate levels.

Patients who received 8 mg of PEG-uricase every two weeks had thegreatest reduction in PUA with levels below 6 mg/dL 92 percent of thetreatment time (pre-treatment plasma urate of 9.1 mg/dL vs. mean plasmaurate of 1.4 mg/dL over 12 weeks).

Substantial and sustained lower plasma urate levels were also observedin the other PEG-uricase treatment dosing groups: PUA below 6 mg/ml 86percent of the treatment time in the 8 mg every four weeks group(pre-treatment plasma urate of 9.1 mg/dL vs. mean plasma urate of 2.6mg/dL over 12 weeks); PUA below 6 mg/ml 84 percent of the treatment timein the 12 mg every four weeks group (pre-treatment plasma urate of 8.5mg/dL vs. mean plasma urate of 2.6 mg/dL over 12 weeks); and PUA below 6mg/ml 73 percent of the treatment time in the 4 mg every two weeks group(pre-treatment plasma urate of 7.6 mg/dl, vs. mean plasma urate of 4.2mg/dL over 12 weeks).

The maximum percent decrease in plasma uric acid from baseline withinthe first 24 hours of PEG-uricase dosing was 72% for subjects receiving4 mg/2 weeks (p equals 0.0002); 94% for subjects receiving 8 mg/2 weeks(p less than 0.0001); 87% for subjects receiving 8 mg/4 weeks (p lessthan 0.0001); and 93% for subjects receiving 12 mg/4 weeks (p less than0.0001).

The percent decrease in plasma uric acid from baseline over the 12-weektreatment period was 38% for subjects receiving 4 mg/2 weeks (p equals0.0002); 86% for subjects receiving 8 mg/2 weeks (p less than 0.0001);58% for subjects receiving 8 mg/4 weeks (p equals 0.0003); and 67% forsubjects receiving 12 mg/4 weeks (p less than 0.0001).

Surprisingly, some subjects receiving PEG-uricase experienced aninfusion related adverse event, i.e., an infusion reaction. Thesereactions occurred in 14% of the total infusions.

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

Many modifications and variations of the present invention can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims along with the full scope ofequivalents to which such claims are entitled.

1-37. (canceled)
 38. A method of reducing elevated uric acid levels in asubject in need thereof, comprising administering a pharmaceuticalcomposition comprising: a conjugate comprising a uricase and monomethoxypolyethylene glycol (mPEG), wherein the uricase comprises the amino acidsequence of SEQ ID NO:8; wherein the mPEG has a molecular weight ofabout 10 kDa; wherein the mPEG is covalently attached to a lysineresidue of the uricase.
 39. The method of claim 38, wherein theconjugate comprises about 2-12 mPEG molecules per uricase monomer. 40.The method of claim 38, wherein the uricase is a tetramer.
 41. Themethod of claim 39, wherein the pharmaceutical composition comprises 8mg of the uricase.
 42. The method of claim 38, wherein thepharmaceutical composition comprises 8 mg of the uricase per mL ofsolution.
 43. The method of claim 38, wherein the pharmaceuticalcomposition further comprises a saline solution.
 44. The method of claim42, wherein the pharmaceutical composition is diluted into 250 mL ofsaline solution for administration.
 45. The method of claim 38, whereinthe pharmaceutical composition is administered intravenously.
 46. Themethod of claim 38, wherein the pharmaceutical composition isadministered at a dosage of 8 mg of the uricase.
 47. The method of claim38, wherein the pharmaceutical composition is administered at a dosageof 8 mg of the uricase every two weeks.
 48. The method of claim 38,wherein the pharmaceutical composition is administered over a 120-minuteperiod.
 49. The method of claim 48, wherein the uric acid level isreduced in the plasma of the subject.
 50. The method of claim 49,wherein the plasma uric acid level is lowered to 6.0 mg/dl or less. 51.The method of claim 49, wherein the subject has a plasma uric acid levelof 6.0 mg/dl or less for at least 80% of a treatment period.
 52. Themethod of claim 47, wherein the subject receives an antihistamine or acorticosteroid prior to the administration of the conjugate.
 53. Themethod of claim 47, wherein the subject receives acetaminophen prior tothe administration of the conjugate.
 54. The method of claim 47, whereinthe subject receives a nonsteroidal anti-inflammatory drug (NSAID) priorto the administration of the conjugate.
 55. The method of claim 47,wherein the subject is an adult subject.
 56. The method of claim 47,wherein the subject is suffering from gout.
 57. The method of claim 47,wherein the subject is suffering from gout that is refractory toconventional therapy.